Bottom Line:
Variable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner.Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control.These data demonstrate that histone acetylation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.

ABSTRACTVariable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner. Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control. Here, we investigate the role for histone acetylation in the developmentally regulated rearrangements of the mouse TCR-gamma gene, wherein predominant rearrangement is switched from Vgamma3 to Vgamma2 gene during the fetal to adult thymocyte development. Our results indicate that histone acetylation correlates with accessibility, as histone acetylation at the fetal-type Vgamma3 gene in accord with germline transcription is relatively high in fetal thymocytes, but specifically becomes low in adult thymocytes within the entirely hyperacetylated locus. Furthermore, inhibition of histone deacetylation during the development of adult bone marrow-derived thymocytes by a specific histone deacetylase inhibitor, trichostatin A, leads to elevated histone acetylation, germline transcription, cleavage, and rearrangement of the Vgamma3 gene. These data demonstrate that histone acetylation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.

Figure 2: TSA induces Vγ3 rearrangements in adult-derived thymocytes. (A) Flow cytometric analysis of organ-cultured thymocytes in fetal thymic lobes repopulated with E14 fetal liver and adult bone marrow (BM) cells. Thymocytes cultured for 14 d with (+) or without (−) TSA were stained with FITC-Vγ2 and PE-CD3ε, or PE-Vγ3 and FITC-CD3ε antibodies. Average cell numbers per lobe from four lobes and percentages of cells for a given phenotype are shown above and inside each panel, respectively. (B) PCR analysis of organ-cultured thymocyte DNA to measure coding joints (CJ) and signal joints (SJ) of Vγ2-Jγ1 or Vγ3-Jγ1, and CD3ε gene for a control. Fivefold serial dilutions of E16 and adult TN (ATN) thymocyte (Thy) DNA and no DNA control (DNA−) were also subjected to PCR.

Mentions:
To address this hypothesis, we tested whether inhibition of histone deacetylation by TSA 22 affects development of Vγ3+ T cells in FTOC (Fig. 2 A). E14 fetal liver and adult bone marrow cells were transferred into 2′-deoxyguanosine–treated fetal thymic lobes and then cultured with or without TSA. After 14 d, thymocytes were prepared from the cultured lobes and analyzed by flow cytometry. Both fetal liver and adult bone marrow cells gave rise to Vγ2+ T cells irrespective of TSA treatment. In contrast, a small but discrete Vγ3+ T cell population was detected in fetal- but not adult-derived thymocytes without TSA, confirming our previous finding that the potential to differentiate into Vγ3+ T cells is determined at the stem cell level 14. However, in the presence of TSA both fetal liver and adult bone marrow cells gave rise to distinct Vγ3+ populations. This is not due to relative enrichment because the absolute numbers of Vγ3+ cells, but not of Vγ2+ cells, were significantly increased by TSA. These data indicate that TSA treatment promotes development of fetal-type Vγ3+ T cells. We also verified the donor origin of Vγ3+ cells by allotypic marker (CD45.1 and CD45.2) staining (data not shown).

Figure 2: TSA induces Vγ3 rearrangements in adult-derived thymocytes. (A) Flow cytometric analysis of organ-cultured thymocytes in fetal thymic lobes repopulated with E14 fetal liver and adult bone marrow (BM) cells. Thymocytes cultured for 14 d with (+) or without (−) TSA were stained with FITC-Vγ2 and PE-CD3ε, or PE-Vγ3 and FITC-CD3ε antibodies. Average cell numbers per lobe from four lobes and percentages of cells for a given phenotype are shown above and inside each panel, respectively. (B) PCR analysis of organ-cultured thymocyte DNA to measure coding joints (CJ) and signal joints (SJ) of Vγ2-Jγ1 or Vγ3-Jγ1, and CD3ε gene for a control. Fivefold serial dilutions of E16 and adult TN (ATN) thymocyte (Thy) DNA and no DNA control (DNA−) were also subjected to PCR.

Mentions:
To address this hypothesis, we tested whether inhibition of histone deacetylation by TSA 22 affects development of Vγ3+ T cells in FTOC (Fig. 2 A). E14 fetal liver and adult bone marrow cells were transferred into 2′-deoxyguanosine–treated fetal thymic lobes and then cultured with or without TSA. After 14 d, thymocytes were prepared from the cultured lobes and analyzed by flow cytometry. Both fetal liver and adult bone marrow cells gave rise to Vγ2+ T cells irrespective of TSA treatment. In contrast, a small but discrete Vγ3+ T cell population was detected in fetal- but not adult-derived thymocytes without TSA, confirming our previous finding that the potential to differentiate into Vγ3+ T cells is determined at the stem cell level 14. However, in the presence of TSA both fetal liver and adult bone marrow cells gave rise to distinct Vγ3+ populations. This is not due to relative enrichment because the absolute numbers of Vγ3+ cells, but not of Vγ2+ cells, were significantly increased by TSA. These data indicate that TSA treatment promotes development of fetal-type Vγ3+ T cells. We also verified the donor origin of Vγ3+ cells by allotypic marker (CD45.1 and CD45.2) staining (data not shown).

Bottom Line:
Variable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner.Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control.These data demonstrate that histone acetylation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.

ABSTRACTVariable/diversity/joining (V[D]J) recombination of the T cell receptor (TCR) and immunoglobulin (Ig) genes is regulated by chromatin accessibility of the target locus to the recombinase in a lineage- and stage-specific manner. Histone acetylation has recently been proposed as a molecular mechanism underlying the accessibility control. Here, we investigate the role for histone acetylation in the developmentally regulated rearrangements of the mouse TCR-gamma gene, wherein predominant rearrangement is switched from Vgamma3 to Vgamma2 gene during the fetal to adult thymocyte development. Our results indicate that histone acetylation correlates with accessibility, as histone acetylation at the fetal-type Vgamma3 gene in accord with germline transcription is relatively high in fetal thymocytes, but specifically becomes low in adult thymocytes within the entirely hyperacetylated locus. Furthermore, inhibition of histone deacetylation during the development of adult bone marrow-derived thymocytes by a specific histone deacetylase inhibitor, trichostatin A, leads to elevated histone acetylation, germline transcription, cleavage, and rearrangement of the Vgamma3 gene. These data demonstrate that histone acetylation functionally determines the chromatin accessibility for V(D)J recombination in vivo and that an epigenetic modification of chromatin plays a direct role in executing a developmental switch in cell fate determination.